Gastrointestinal absorption appears to be the most desirable route for the administration of drugs including therapeutic peptides and proteins, because it can reduce both the inconvenience and the complications associated with injection, and increase patient compliance especially in chronic administration. However, no acceptable oral formulation is currently available for peptide and protein drugs. In this application, a novel approach, which is based on our preliminary findings of the transferrin receptor-mediated transcytosis in intestinal epithelial cells, will be explored for increasing peptide and protein absorption in the gastrointestinal tract. Our hypothesis is that there is a constitutive transcytosis of transferrin receptors from the luminal to the basolateral membranes in the gastrointestinal tract due to an alternative pathway for the membrane sorting in intestinal epithelial cells. The advantages of this approach include the specificity and the stability of transferrin, as well as the abundance of transferrin receptors in the intestinal epithelium. Transferrin conjugates of insulin and granulocyte colony stimulating factor (G-CSF) will be prepared. Both reversible and irreversible linkages for the conjugation will be investigated. In order to obtain structurally defined conjugates, transferrin with a single modification site will be produced by two different approaches; by purification using biotin-avidin affinity chromatography, and by recombinant technology to generate a mutagenized transferrin containing a single free sulfhydryl group for conjugation. Insulin-transferrin and G-CSF-transferrin conjugates will be first characterized by using biochemical and biological methods, including PAGE, immunoassays, transcytosis in human enterocyte- like Caco-2 cells, and in vitro bioassays. Selected conjugates will be administered to rats or mice, both subcutaneously and orally, to measure hypoglycemic and neutrophilic activities for insulin and G-CSF, respectively. Tyrphostin 8, a specific enhancer for transferrin receptor-mediated transcytosis, will be co-administered to evaluate the increase of the transport efficiency. This proposal will provide essential information for the development of either chemically synthesized or genetically engineered protein-transferrin conjugates as safe and effective drugs that can be administered orally for the treatment of various human diseases.